Distinctive cognate sequence discrimination, bound DNA conformation, and binding modes in the E2 C-terminal domains from prototype human and bovine papillomaviruses

The C-terminal DNA binding domain of the E2 protein is involved in transcri ptional regulation and DNA replication in papillomaviruses. At low ionic st rength, the domain has a tendency to form aggregates, a process readily rev ersible by the addition of salt. While fluorescence anisotropy measurements show a 1:1 stoichiometry at pH 5.5, we observed that a second HPV-16 E2 C- terminal dimer can bind per DNA site at pH 7.0. This was confirmed by displ acement of bis-ANS binding, tryptophan fluorescence, native electrophoresis , and circular dichroism. The two binding events are nonequivalent, with a high-affinity binding involving one E2C dimer per DNA molecule with a K-D o f 0.18 +/- 0.02 nM and a lower affinity binding mode of 2.0 +/- 0.2 nM. The bovine (BPV-1) E2 C-terminal domain binds to an HPV-16 E2 site with 350-fo ld lower affinity than the human cognate domain and binds 7-fold less tight ly even to a bovine-derived DNA site. The ability to discriminate between c ognate and noncognate sequences is 50-fold higher for the human domain, and the latter is 180-fold better than the bovine at discriminating specific f rom nonspecific DNA. A substantial conformational change in bound DNA is ob served by near-UV circular dichroism. The bovine domain imposes a different DNA conformation than that caused by the human counterpart, which could be explained by a more pronounced bent. Structure-function differences and bi ochemical properties of the complexes depend on the protein domain rather t han on the DNA, in line with crystallographic evidence. Despite the strong sequence homology and overall folding topology, the differences observed ma y explain the distinctive transcriptional regulation in bovine and human vi ruses.